Coordinated Cytokinin Signaling And Auxin Biosynthesis Mediates Arsenate-induced Root Growth Inhibition

Arsenic pollution in groundwater and soil is one of the biggest challenges of the modern world,and it severely restricts agricultural development and threatens human health.Recent research focused on understanding the molecular mechanisms,which allow plants to uptake,transport,and detoxification of As,but the mechanism responsible for As inhibition of root growth has received little or no attention.Root architecture plays important roles in plant water and nutrient acquisition,making the plasticity of root growth an important adaptive trait.Interactions between plant hormones and environmental signals are important for the maintenance of root growth plasticity under ever-changing environmental conditions.Through genetics,cell biology,biochemistry and other biological methods,our results reveal cytokinin and auxin co-regulating the molecular mechanism of Arabidopsis root growth under arsenic stress.And the main results are as follows:(1)As~V inhibits elongation of the primary root.We examined the effect of different concentrations of As~V on the primary root length of Col-0 seedlings,and found that the growth of Col-0 roots was inhibited by As~V in a dose-dependent manner.Further,closer observation of the DZ and EZ of As~V-treated roots indicated that As~V reduced both the number and length of cells in these regions.Meanwhile,As~V inhibits the expression of pro CYCB1;1::GUS,these data indicate that As~V reduces both cell proliferation and cell elongation.As~V induces ASA1 and ASB1 expression,suggesting that As~V promotes auxin biosynthesis by activating ASA1 and ASB1 transcription.(2)ASA1-and ASB1-mediated auxin biosynthesis is involved in As~V-induced root growth inhibition.RNA-seq data indicates that auxin is involved in early As~V-induced root growth inhibition;it is further found that As~V induces the expression of the auxin marker DR5::GUS,and at the same time promotes the biosynthesis of endogenous auxin in the root tip.asa1 asb1showed more insensitivity to As~V than Col-0 under As~V stress.All these lines of evidence demonstrate that As~V-induced root growth inhibition is partially dependent on ASA1-and ASB1-mediated auxin biosynthesis.(3)ARR1 promotes ASA1 and ASB1 expression through promoter binding.We performed Y1H screening using Arabidopsis c DNA libraries harboring the HIS3reporter gene under the control of the ASA1 promoter,and screened type-B Arabidopsis response regulators ARR1 and ARR10.Electrophoretic mobility shift assays(EMSA)and chromatin immunoprecipitation(Ch IP)assays confirm that ARR1 directly binds to ASA1 and ASB1 promoters by binding CTTCGATCTT,GAGGATTGTC and TTCGATCCAT sites in vitro and in vivo.Next,Nicotiana benthamiana transient expression assays confirm that ARR1 directly activates ASA1 and ASB1 expression in vivo.The results of q RT-PCR proved that the expression of ASA1 and ASB1 induced by As~V is dependent on ARR1 and ARR10.(4)ARR proteins play an important role in As~V-induced root growth inhibition.Because of the redundancy of type-B ARR transcription factors,roots of arr1-3,arr10-5,and arr12-1 single mutant showed similar root growth inhibition rate as that of Col-0 roots in the presence of As~V.However,compared with that of Col-0 seedlings,the roots of arr1-3arr10-5 and arr1-3 arr12-1 seedlings showed significantly reduced sensitivity to As~V and root lengths of ARR1-SRDX and ARR10-SRDX transgenic lines showed reduced sensitivity to As~V.These data indicate that As~V-mediated root growth inhibition is partially dependent on ARR1and ARR10.As~V induces cytokinin synthetic sensor TCSn::GFP expressed in root caps,demonstrating that As~V activates cytokinin signaling.We used transgenic Arabidopsis lines ARR1/10/12-C1-Ypet,which reflect the nearest endogenous expression and localization of ARRs,to monitor the cellular distribution patterns of ARRs proteins under As~V stress.Addition of the MG132 or As~V separately to the transgenic seedlings led to increased intensity of fluorescence,and the fluorescence with MG132 was much stronger than that with As~V.Co-treatment with MG132 and As~V showed similar fluorescence intensities with MG132treatment,these supported the As~V-induced accumulation of ARR proteins in root tips by repressing the activity of 26S proteasome.(5)Both ASA1 and ASB1 act genetically downstream of ARR1 to regulate As~V-induced root growth inhibition.Genetic analysis revealed ASA1 and ASB1 act downstream of ARR1 in the root growth inhibition by As~V.More importantly,TCSn::GFP was activated earlier than DR5rev:GFP in the presence of As~V.Roots of aux1 and pin2 mutant seedlings showed reduced sensitivity to As~Vstress.AUX1 and PIN2 expression in root tips was not regulated by As~V which implies that basipetal auxin transport may not be affected by As~V stress.Additionally,DII-VENUS was significantly downregulated in the root meristem and EZ by As~V in ARR1-and ARR10-dependent manner.These data demonstrate that As~V stress induces auxin synthesis in root tips by activating cytokinin signals,and excess auxin accumulation in the EZ due to AUX1-and PIN2-mediated basipetal transport from root tips to EZ inhibits cell elongation in the EZ,which leads to primary root growth arrest.